Structure-based optimization, in vitro profiling, and ADMET prediction of Celastrol derivatives as potent prolyl...

Prolyl endopeptidase (PREP) is a potential therapeutic target for neurological disorders. Through screening a triterpenoid compound library, this study identified celastrol as a novel PREP inhibitor (IC₅₀ = 33.35 μM). Furthermore, among three derivatives designed and synthesized based on this lead compound, Ce-2 (C3-acetylated) and Ce-3 (C3-butyrylated) showed significantly enhanced activity, with IC₅₀ values of 4.46 μM and 3.89 μM, respectively, representing 7.5-fold and 8.6-fold improvements compared to celastrol. Kinetic studies demonstrated that both function as mixed-type inhibitors (Kᵢ values of 4.40 μM and 4.17 μM, respectively). Molecular docking suggested their putative binding modes: Ce-2 formed polar contacts with Asn477, Ser554, and His680 and contacted Arg643 in the S2 subsite, while Ce-3 formed additional polar interactions involving Arg643, Asn477, and Asp149. Subsequent molecular dynamics simulations indicated that both compounds remained accommodated within the PREP binding cavity but exhibited distinct interaction profiles: Ce-3 maintained frequent contacts with several canonical pocket residues, whereas Ce-2 lacked persistent direct contacts under the applied analysis criteria, indicating that its detailed binding mechanism requires further clarification. AutoDock Vina yielded docking scores of -7.8 and - 7.9 kcal/mol for Ce-2 and Ce-3, respectively, supporting their putative binding modes within the PREP cavity. ADMET analysis identified both favorable parameters and important developability limitations, and cellular experiments confirmed that both compounds effectively inhibit endogenous PREP activity in neuronal cells in a concentration-dependent manner without significant cytotoxicity under the tested conditions. This study identified Ce-2 and Ce-3 as early-stage lead-like compounds, providing a foundation for further optimization of celastrol-derived PREP inhibitors.